PROJECT SUMMARY Tumor-induced immunosuppressive factors hamper tumor clearance by activated immune cell and significantly contribute to metastasis; this has driven the development of immune modulatory drugs to re-activate immune cells. Using an immune competent murine model of human papillomavirus positive (HPV+) head and neck squamous cell carcinoma (HNSCC) we found that tumors over-expressing the signaling ligand, EphrinB1, induce increased metastasis and harbor significantly more suppressor FoxP3 + T regulatory cells. These data suggest that the interaction between tumor-expressed EphrinB1 and immune cell-expressed Eph receptors (cognate Ephrin receptors) induces immune suppression. We hypothesize that tumor expressed signals, including EphrinB1, contribute to metastasis by modulating immune cell function and dampening anti- tumor immunity. To test this hypothesis, we propose the following specific aims as follows. In Aim 1, we will define how tumor-derived EphrinB1 influences protective vs. tumor-promoting T cells. Using a combination of Eph knock-out mice together with flow cytometry, we will identify the critical T cell (CD4+ or CD8+) as well as the key Ephrin and Eph components that induce immune suppression. Should we find that T cells do not significantly contribute to tumor tolerance, alternative experiments will address the potential role of myeloid derived suppressor cells (MDSCs). In Aim 2 we will define the contribution of Ephrin and Eph signaling to metastasis in human cancer. According to The Cancer Genome Atlas, a high percent of prostate and breast cancer cases harbor amplifications of either Ephrin ligands or Eph receptors. Thus, blood from these cancer patients will be functionally analyzed for immune suppression (T regulatory cells and possibly MDSCs) and correlated with patient metastasis and survival data. At the completion of this project, the contribution of tumor-expressed EphrinB1 to immune cell function and metastasis will not only be defined but the mechanism determined.These data will provide targets for therapeutic intervention to block immune suppression and metastasis.